Photosynthesis

Mechanisms for Membrane Protein
Crystallization: Analysis by Small-Angle
Neutron Scattering

The goal of this project is to characterize mechanisms for
crystallization of membrane proteins, using neutron and X-ray
scattering techniques. Intrinsic membrane proteins are proteins
that are localized within biological lipid membranes, and they
are responsible for major energy-converting biological
processes, such as photosynthesis, respiration, vision, and
nerve conduction. Membrane protein crystallization is complicated
by the requirement for the use of solubilizing detergent to replace
the natural membrane in crystallization mixtures. The particle to
be crystallized consists of a heterogeneous, protein-detergent
complex. Progress occurred in two areas in FY98 that focused
on determining the role of the detergent in the crystallization process.

First, the effect of detergent alkyl chain length on the crystallization
of detergent-solubilized membrane proteins was characterized
using the reaction center from Rhodobacter sphaeroides as a
model. Crystallization was performed in the presence of both
n-alkyl-b-D-glucopyranosides, C_nG, where the alkyl chain length,
n, was varied from 8 to 10, and dodecyl-dimethylamine-N-oxide
(C₁₂DAO). A pronounced effect of detergent chain length was
found on the ability to crystallize the reaction center. Small angle
neutron scattering measurements (SANS) demonstrated that the
variation in the ability to crystallize the reaction center is correlated
with changes in the apparent radius of gyration, R_g, of the reaction
center-detergent particle, reflecting changes in the size of the
reaction center particle and strength of the particle-particle
interactions. Specifically, reaction center crystallization in the
absence of additional amphiphiles was found to occur only with
octylglucoside, C₈G. Crystallization in the presence of longer
chain detergents, C₉G, C₁₀G, C₁₂DAO, required the use of
an additional small amphiphile, heptanetriol (HT). SANS
measurements showed that the apparent R_g of the reaction
center-detergent particle increased with increasing detergent
chain length, and that the addition of HT reduces this parameter.
The addition of HT was found to impede crystallization in the
presence of C₈G. This inhibition could be reversed by increasing
C₈G concentration with respect to HT, resulting in the formation
of alternate crystal forms. Taken together, these results suggest
that detergents and small-molecule amphiphiles used in membrane
protein crystallization must be chosen to optimize both the size
and solubility of the protein-detergent particle. Data for the
reaction center suggest that crystallization occurs within a restricted
range of apparent R_g.

Significant progress also occurred in a second area, in the
development of experimental and theoretical procedures for using
"multidimensional" scattering measurements to analyze
macromolecular structures in solution. This approach involves
simultaneous measurement of small and wide angle X-ray,
anomalous X-ray, and neutron scattering of native and isotopically
enriched protein/detergent assemblies. The use of multiple
scattering measurements with isotopic labeling is essential for
recovering structural information lost in typical solution scattering
experiments. We demonstrated the value of this multidimensional
approach by resolving not only the overall size and shape of the
C_nG and C₁₂DAO micelles in solution, but by also resolving
details of micelle fine structure as functions of alkyl chain length,
concentration, and solution conditions. X-ray scattering data
were found to reflect contributions from the complete micelle
core-shell structure, whereas the neutron data reflected a uniform
scattering density distribution primarily from the alkane core.
Numerical modeling of both X-ray and neutron scattering data
found that C_nG micelle shape could be best fit with a cylindrical
form factor, with dimensions dependent upon the surfactant
concentration and alkyl chain length. The micelle length was
found to increase dramatically with increases in surfactant chain
length, presumably reflecting imperfect packing of the surfactant
in the cap regions and solvent exposure of the alkyl chains.
X-ray scattering intensity was found to decay more slowly as
a function of scattered angle than could be explained by a
simple core-shell structure. The anomalous decay of scattered
intensity at wide angles was fit by modeling fluctuations in the
position of surfactant molecules within the equilibrium core-shell
structure, in a manner analogous to the use of a Debye-Waller
temperature factor in crystallographic data analysis. These
findings suggest that measurement of wide angle X-ray and
neutron scattering provides the means to measure amplitudes
of surfactant disorder at the micelle-aqueous interface, and that
these fluctuations may contribute significantly to the
physical-chemical activity of these surfactants in crystallization
applications.

Contact: D. M. Tiede

Return to Photosynthesis

Glassblowing

Interfacial Processes

Radiation and Photochemistry

Photosynthesis
Biological Materials Growth Facility

Cluster Studies

Chemical Dynamics

Atomic Physics

Nanophotonics

Heavy Elements

Coordination Chemistry

f-Electron Interactions

Actinide Facility

Computational Materials and Electrochemical Processes